Bottom Line:
However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness.Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness.We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

ABSTRACTA method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

Mentions:
As discussed, a primary motivation for developing far-red FPs is to perform deep-tissue imaging in mammals using excitation wavelengths above 600 nm, which penetrate through hemoglobin-rich tissues more easily than bluer light16. To directly compare the imaging performance of far-red FPs in deep tissue, we first imaged purified far-red proteins placed within the esophagus of an euthanized mouse using a commercial small-animal fluorescence imaging system. Imaged from the ventral side, this location is approximately 7 mm deep. With 605/30 nm excitation light (center wavelength/full width at half-maximum), a range that covers the excitation peaks of all tested FPs, mNeptune2.5 was the brightest (Fig. 3a), consistent with mNeptune2.5 having the highest peak brightness by spectroscopy (Table 1). When using 640/30 nm light, mCardinal was uniquely brighter than all other far-red FPs (Fig. 3b), consistent with its predicted advantage at these redder excitation wavelengths (Table 1). We also compared mCardinal with iRFP, a bacteriophytochrome mutant that binds biliverdin to enhance its infrared fluorescence, for the ability to detect gene expression in the liver following hydrodynamic injection of plasmid DNA. Compared to iRFP excited at 675/30 nm, mCardinal yielded a higher signal/background ratio with 605/30 nm excitation and a similar signal/background ratio with 640/30 nm excitation (Supplementary Fig. 12). In preparation for μm-resolution imaging in mouse tissues, we equipped a fluorescence stereoscope using 620/20 nm excitation filters, selected so that all excitation wavelengths are beyond 600 nm while still efficiently exciting far-red FPs. In this system, mCardinal again yielded the highest brightness among the mNeptune derivatives (Fig. 3c). Thus, mCardinal performs especially well in deep-tissue imaging with wavelengths of light above 600 nm.

Mentions:
As discussed, a primary motivation for developing far-red FPs is to perform deep-tissue imaging in mammals using excitation wavelengths above 600 nm, which penetrate through hemoglobin-rich tissues more easily than bluer light16. To directly compare the imaging performance of far-red FPs in deep tissue, we first imaged purified far-red proteins placed within the esophagus of an euthanized mouse using a commercial small-animal fluorescence imaging system. Imaged from the ventral side, this location is approximately 7 mm deep. With 605/30 nm excitation light (center wavelength/full width at half-maximum), a range that covers the excitation peaks of all tested FPs, mNeptune2.5 was the brightest (Fig. 3a), consistent with mNeptune2.5 having the highest peak brightness by spectroscopy (Table 1). When using 640/30 nm light, mCardinal was uniquely brighter than all other far-red FPs (Fig. 3b), consistent with its predicted advantage at these redder excitation wavelengths (Table 1). We also compared mCardinal with iRFP, a bacteriophytochrome mutant that binds biliverdin to enhance its infrared fluorescence, for the ability to detect gene expression in the liver following hydrodynamic injection of plasmid DNA. Compared to iRFP excited at 675/30 nm, mCardinal yielded a higher signal/background ratio with 605/30 nm excitation and a similar signal/background ratio with 640/30 nm excitation (Supplementary Fig. 12). In preparation for μm-resolution imaging in mouse tissues, we equipped a fluorescence stereoscope using 620/20 nm excitation filters, selected so that all excitation wavelengths are beyond 600 nm while still efficiently exciting far-red FPs. In this system, mCardinal again yielded the highest brightness among the mNeptune derivatives (Fig. 3c). Thus, mCardinal performs especially well in deep-tissue imaging with wavelengths of light above 600 nm.

Bottom Line:
However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness.Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness.We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

ABSTRACTA method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.